PMID- 10649434
OWN - NLM
STAT- MEDLINE
DCOM- 20000320
LR  - 20151119
IS  - 0730-2312 (Print)
IS  - 0730-2312 (Linking)
VI  - 76
IP  - 3
DP  - 2000 Jan
TI  - Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces
      its GDP/GTP exchange rate.
PG  - 368-75
AB  - The Galpha subunits of heterotrimeric G proteins are constituted by a conserved
      GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon
      GTP binding, four regions, called switch I, II, III, and IV, have been identified
      as undergoing structural changes. Switch I, II, and III are located in RasD and
      switch IV in HD. All Galpha known functions, such as GTPase activity and
      receptor, effector, and Gbetagamma interaction sites have been found to be
      localized in RasD, but little is known about the role of HD and its switch IV
      region. Through the construction of chimeras between human and Xenopus Gsalpha we
      have previously identified a HD region, encompassing helices alphaA, alphaB, and 
      alphaC, that was responsible for the observed functional differences in their
      capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett
      340:249-254). Since switch IV is located within this region and contains most of 
      the nonconservative amino acid differences between both Gsalpha proteins, in the 
      present work we constructed two human Gsalpha mutant proteins in which we have
      changed four and five switch IV residues for the ones present in the Xenopus
      protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17
      (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia
      coli, purified, and characterized by their ability to bind GTPgammaS, dissociate 
      GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP
      release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17
      having considerably slower kinetics than M15 for all functions tested.
      Reconstituted adenylyl cyclase activity with both mutants showed normal
      activation in the presence of AlF(4)(-), but a decreased activation with
      GTPgammaS, which is consistent with the lower GDP dissociating rate they
      displayed. These data provide new evidence on the role that HD is playing in
      modulating the GDP/GTP exchange of the Gsalpha subunit.
CI  - Copyright 2000 Wiley-Liss, Inc.
FAU - Echeverria, V
AU  - Echeverria V
AD  - Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Casilla
      160-C, Universidad de Concepcion, Concepcion, Chile.
FAU - Hinrichs, M V
AU  - Hinrichs MV
FAU - Torrejon, M
AU  - Torrejon M
FAU - Ropero, S
AU  - Ropero S
FAU - Martinez, J
AU  - Martinez J
FAU - Toro, M J
AU  - Toro MJ
FAU - Olate, J
AU  - Olate J
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Cell Biochem
JT  - Journal of cellular biochemistry
JID - 8205768
RN  - 0 (DNA Primers)
RN  - 0 (Recombinant Proteins)
RN  - 146-91-8 (Guanosine Diphosphate)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - EC 3.4.21.4 (Trypsin)
RN  - EC 3.6.5.1 (GTP-Binding Protein alpha Subunits, Gs)
RN  - EC 4.6.1.1 (Adenylyl Cyclases)
SB  - IM
MH  - Adenylyl Cyclases/metabolism
MH  - Base Sequence
MH  - DNA Primers/genetics
MH  - GTP-Binding Protein alpha Subunits, Gs/chemistry/*genetics/*metabolism
MH  - Gene Expression
MH  - Guanosine Diphosphate/metabolism
MH  - Guanosine Triphosphate/metabolism
MH  - Humans
MH  - In Vitro Techniques
MH  - Models, Molecular
MH  - Mutagenesis, Site-Directed
MH  - Protein Binding
MH  - Protein Structure, Secondary
MH  - Protein Structure, Tertiary
MH  - Recombinant Proteins/chemistry/genetics/metabolism
MH  - Trypsin
EDAT- 2000/01/29 09:00
MHDA- 2000/03/25 09:00
CRDT- 2000/01/29 09:00
PHST- 2000/01/29 09:00 [pubmed]
PHST- 2000/03/25 09:00 [medline]
PHST- 2000/01/29 09:00 [entrez]
AID - 10.1002/(SICI)1097-4644(20000301)76:3<368::AID-JCB4>3.0.CO;2-B [pii]
PST - ppublish
SO  - J Cell Biochem. 2000 Jan;76(3):368-75.